Biotechnological methods are used to an increasing extent in the production of proteins, peptides, nucleic acids and other biological molecules and compounds, for research purposes as well as in order to prepare novel kinds of drugs. Due to its versatility and sensitivity to the compounds, liquid chromatography is often the preferred purification method in this context. The term liquid chromatography embraces a family of closely related separation methods, which are all based on the principle that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile (liquid) phase, which is contacted with a stationary phase.
The target compound will then undergo a series of interactions between the stationary and mobile phases as it is being carried through the system by the mobile phase. In brief, the concept of chromatography relates to the separation of target molecules from other molecules in a sample based on differences in their respective physical or chemical properties in relation to the mobile and the stationary phases.
Examples of commonly used chromatography purification techniques includes, but is not limited to: affinity chromatography (AC), Immobilized metal ion affinity chromatography (IMAC), flow-through chromatography, ion exchange chromatography (IEX), size-exclusion chromatography, reversed-phase chromatography (RPC), simulated moving-bed chromatography, hydrophobic interaction chromatography (HIC), gel filtration (GF), chromato-focusing and the like. Sometimes a purification protocol includes two or more purification steps of the using the same or different purification techniques.
The volume of the collected fractions is often different during different steps in a chromatographic run. During sample application, larger fraction volumes are collected as a safety measure in case the target protein was to pass straight through the column. The flow-through is collected in one or a few fractions corresponding to the volume of the sample applied and the subsequent wash. During elution, smaller fraction volumes are usually collected, and an eluting peak is normally divided into a number of eluent fractions in order to obtain pure protein from overlapping peaks.
To be able to analyze different parts of the peak, the fraction size during elution is usually set to a value smaller than the expected peak volume. When straight (sometimes called fixed) fractionation is used, the fraction collector will continuously switch tubes according to the set volume throughout the entire fractionation, as shown in FIG. 1a. To further increase the purity of the collected protein peaks, “peak fractionation” can be used. The UV/Vis detector is then used to determine when to start and stop peak fractionation, as shown in FIG. 1b. Straight fractionation and peak fractionation can also be combined during a run.
The fractionation delay volume is the volume between the UV/Vis detector's flow cell and the fraction collector. It is important that the correct delay volume is entered in the software.
The defined delay volume will be used by the system to calculate the time T1, which is when the peak reaches the fraction collector. T1 is used to synchronize the fractionation marks in the chromatogram with the tube switch of the fraction collector (see FIG. 2). At the start of the fraction collection, the delay volume is directed to waste or the first fractionation tube depending on which system is used. The delay volume depends on the tubing and components included in the flow path, and the delay volume is determined theoretically or experimentally by including the volume from all tubing and components between the absorbance detector and the fractionation tip.
Analytic peak integration and preparative peak integration of samples containing one or more target molecules using liquid chromatography are well known in the field. During analytic peak integration, or analytic run, the total amount of or ratio between one or more target molecules in the sample is determined by estimating the full peak relating to each target molecule and integrating said peak. During preparative peak integration, or preparative run, the object is to collect as pure portions of one or more molecules as possible.
Traditionally, the user has been forced to decide the type of peak integration before the sample is purified. If analytic peak integration is selected, an integration window is selected and a baseline established. Different techniques can be used to establish the baseline and also to identify the peaks within the integration window.
In preparative peak integration, the eluent flow (i.e. the output liquid flow of purified sample from the liquid chromatography purification) is divided into eluent fractions that are collected after the preparative run. Output parameter data is measured by the detector, the most common type is UV absorbance detector, and the data is displayed as a curve, also known as a chromatogram. Each peak is defined in the same way as for analytic peak integration. The user may manually selects which eluent fractions within the identified peak that are interesting to investigate, i.e. that contain the target molecule in an appropriate amount.